Resolution and Reconstitution of the Mitochondrial Electron Transport System

Abstract

Abstract 1. Reconstitution of a complex of succinoxidase from individual partially purified components of the respiratory chain required the time-dependent interaction between all components except succinate dehydrogenase. Succinate dehydrogenase added after formation of the complex interacted rapidly and within seconds succinoxidase activity appeared. 2. An active succinoxidase was formed when cytochrome c and cytochrome oxidase were added after formation of the complex, but the assembly of these components did not correspond to the physiological patterns. Only when these components were present from the beginning, was the oxidation of succinate by the complex resistant to an antiserum against cytochrome c, a response characteristic for phosphorylating submitochondrial particles. 3. A new soluble protein factor was found to be required for the operation of the succinoxidase complex. The factor was retained on a Sephadex G-100 column and was separated from cytochrome c1. It appears to participate in electron transfer between cytochromes b and c1. 4. NADH oxidase was reconstituted by addition of a NADH-coenzyme Q reductase preparation (Complex I) to the succinoxidase complex. Electron micrographs of this NADH oxidase revealed particles resembling submitochondrial particles.